Ligand-targeted emulsions carrying bioactive agents

ABSTRACT

A composition for use in delivering a bioactive agent to targeted tissues or cells comprises: (a) a site-specific targeting ligand; (b) a lipid encapsulated oil in water emulsion; and (c) a bioactive agent in or on the surface of the outer monolayer of the emulsion; the ligand being conjugated directly or indirectly to the emulsion and the composition providing facilitated delivery of the bioactive agent through prolonged association and increased contact of the ligand-bound lipid encapsulated emulsion particles with the lipid bilayer of the target tissues or cells. The composition may also comprise a lipid encapsulated oil in water emulsion and a combination site-specific targeting ligand/bioactive agent. Methods for improved delivery of a bioactive agent to targeted tissues or cells are also disclosed.

BACKGROUND OF THE INVENTION

[0001] This invention relates to ligand-targeted emulsions thatincorporate biologically active agents on or in their particle surface,and more particularly, to such novel emulsions that are especiallyuseful for the treatment of disease with bioactive agents that haveimproved risk/benefit profiles when applied specifically to selectedcells, tissues or organs.

[0002] As used herein, the following terms have the definitions setforth:

[0003] Direct conjugation of ligand to the emulsion particle refers tothe preparation of a ligand-particle complex before administrationwherein the ligand is either adsorbed through ionic, electrostatis,hydrophobic or other noncovalent means to the particle surface (e.g.acylated-antibody), or chemically linked to the surface through covalentbonds to a component of the lipid surface such as a “primer material”(e.g. thio-ether or ester bond), or intrinsically incorporated into thelipid surfactant membrane as a component of the membrane (e.g. a lipidderivatized to a peptiodomimetic agent).

[0004] Indirect conjugation refers to the use of avidin biotin where thecomplex is formed in vivo in two or more steps. An example would begiving the biotinylated antibody first, followed by avidin, and followedby the biotinylated emulsion particle. Any other sequential multistepchemical linking system that could be utilized in vivo is envisioned toproduce the same end result, i.e. the close and specific apposition ofthe emulsion particle to a targeted cell or tissue surface.

[0005] Primer material refers to any constituent or derivatizedconstituent incorporated into the emulsion lipid surfactant layer thatcould be chemically utilized to form a covalent bond between theparticle and a targeting ligand or a component of the targeting ligand(if it has subunits).

[0006] Prolonged association of the emulsion particle with the surfaceof the targeted cell or tissue is in contradistinction to the transientinteraction that an unbound particle, existing free in extracellularbody fluids, would achieve. By binding the particle to the cell surface,the continued circulation of the nanoparticle through the body ishalted. The affixed particle is able to interact with the target cellsurface over an extended period of time. The exact amount of time may bevariable, but is meant to exceed that of more transient nontargetedcontact between particles and cell surfaces by orders of magnitude.

[0007] Surfactant is a term derived from SURFace ACTive AgeNT. ASurfactant is a compound that contains a hydrophilic and a hydrophobicsegment. When added to water or solvents, a surfactant reduces thesurface tension of the systems for the following purposes emulsifying ordispersing in the present application. Our preferred surfactants arephospholipids and cholesterol but include those lipids that arementioned in our previous application and the additional detergentsspecified in our invention disclosure.

[0008] Ligand is a molecule that binds to another molecule, used in thisapplication to refer to a small targeting molecule that bindsspecifically to another molecule on a biological surface separate anddistinct from the emulsion particle itself. The reaction does notrequire nor exclude a molecule that donates or accepts a pair ofelectrons to form a coordinate covalent bond with a metal atom of acoordination complex.

[0009] Emulsion technology is very old and distinct from the more modernliposome technology. This is exemplified by the prolific research andpatent literature involving liposomes since the 1963 report by Bangham(Physical structure and behavior of lipids and lipid enzymes., Adv LipidRes, 1963; 1:65-104). Bangham originally characterized emulsions as“either temporary or permanent dispersions of oils or hydrophobicmaterial in water or vice versa” and liposomes as “ . . . ‘myelins’ and‘myelinics’ . . . irrevocably associated with the structures obtainedwhen certain phospholipids are dispersed in water. . . . The unitstructure is a biomolecular tube of lipids, separated from its adjacentconcentric tube by a layer of water.” In later years liposomes have beenelegantly described as “vesicles in which an aqueous volume is entirelyenclosed by a membrane composed of lipid molecules . . . (which) formspontaneously when these lipids are dispersed in aqueous media. . . .The liposome membrane forms a bilayer structure which is in principleidentical to the lipid portion of natural cell membranes.” Liposomes maybe prepared by a variety of techniques and have single or multiplemembrane layers. They are distinctly different and more complex thanemulsions.

[0010] Drugs can be incorporated into liposomes within either theinternal aqueous phase or within one or more of the lipid bilayermembranes and liposomes can be coupled to ligands of various types.Because of the bilayer nature of a liposome membrane, lipophilic drugsincorporate into both the inner and outer leaflets of the bilayer.Drugs, bound to the inner leaflet layer are unavailable for immediatedelivery by contact facilitated delivery as opposed to lipidencapsulated emulsions. For multilamellar liposomes, most of the drugwill be internalized within the liposome and not readily available forcontact facilitated delivery to a target cell. To extend circulatoryhalf-life, liposomes have been modified with polymerized lipids or theaddition of polyethylene glycol to enhance in vivo survivability. Bothmodifications protect the particles from lipid exchange with other cellsand lipoproteins.

[0011] “An emulsion is a heterogeneous system, consisting of at leastone immiscible liquid intimately dispersed in another in the form ofdroplets, whose diameters, in general, exceed 0.1μ. Such systems possessa minimal stability, which may be accentuated by such additives assurface-active agents, finely-divided solids, etc.” (Becher P. Emulsion:Theory and Practice, New York, N.Y.; Reinhold Publishing Corporation;1965) “The phase which is present in the form of finely divided dropletsis called the dispersion or internal phase; the phase which forms thematrix in which these droplets are suspended is called the continuous orexternal phase. . . . Surface active or other agents which are added toincrease stability . . . are known as emulsifiers or emulsifying agents.Stability is also increased by mechanical devices such as simplestirrers, homogenizers or colloid mills.”

[0012] Liquid perfluorocarbon emulsions are specialized formulationswith various medical and oxygen transport applications. They areespecially useful medically as contrast media, for various biologicalimaging modalities such as nuclear magnetic resonance, ultrasound,x-ray, computed tomography, F-magnetic resonance imaging, and positionemission tomography, as oxygen transport agents or “artificial bloods,”in the treatment of heart attack, stroke, and other vascularobstructions, as adjuvants to coronary angioplasty and in cancerradiation treatment and chemotherapy. The fluorocarbon emulsion can beused to deliver drugs and medicines soluble in or transportable by theemulsion.

[0013] Long et al. U.S. Pat. No. 4,987,154 discloses that fluorocarbonemulsions can deliver therapeutic agents, medicines and drugs throughoutthe body, tissue and organs by at least two modes: 1) within thefluorocarbon phase or 2) by complexing of the agent, medicine or drugwith the surfactant membrane. Long et al. cite examples of medicines,drugs and therapeutic agents that can be dissolved in the fluorocarbonincluding diazepam, cyclosporin, rifampin, clindamycin, isoflurane,halothane and enflurane. Examples of medicines, therapeutic agents anddrugs that do not dissolve in fluorocarbon, but can be complexed with,for example, a lecithin membrane include mannitol, tocopherol,streptokinase, dexamethasone, prostaglandin E, interleukin, gentamycinand cefoxitin. Antibiotics may be delivered transcutaneously through theskin when added to a fluorocarbon emulsion. Furthermore, proteins suchas thrombolytic agents, hormones or enzymes can be transported anddelivered by fluorocarbon emulsions.

[0014] Delivery of drugs as described by Long et al. and others dependupon the encapsulated drug being more slowly metabolized and eliminatedfrom the circulation than free drug. In other cases, the encapsulatedparticles are sequestered into organs and cells involved with the normalmetabolism and clearance of particles and foreign matter from the body,a process referred to as passive targeted delivery. The opportunity toconjugate ligands to perfluorocarbon emulsions for the purpose ofcontact facilitated delivery of bioactive agents was not envisioned byLong et al.

[0015] We have previously reported a novel ligand-targeted,lipid-encapsulated nongaseous perfluorocarbon emulsion useful forultrasound, magnetic resonance and nuclear imaging applications (U.S.Pat. Nos. 5,690,907, 5,780,010, 5,958,371 and 5,989,520). Theperfluorocarbon emulsion is produced through microfluidizationtechniques, and is robustly stable to handling, pressure, atmosphericexposure, heat and shear. In the early phases of development, we coupleda pretargeted biotinylated ligand to a biotinylated version of theemulsion nanoparticle through avidin-biotin interactions. Subsequently,we adopted a direct ligand conjugation approach using monoclonalF_((ab)) fragments to facilitate future clinical implementations.

[0016] The emulsion nanoparticles have long circulatory half-lives dueto their small size and inherent in vivo stability without furthermodification of their outer lipid surfaces with polyethylene glycol orincorporation of polymerized lipids. Surfactant modifications oftendetract from targeting efficacy in order to extend circulatorypersistence. The in vivo clearance of the nanoparticles was measured indogs by quantification of the blood perfluorocarbon content with anestimated half-life of one hour. Preliminary data suggest that thisnovel agent will persist bound to tissue for hours, and dependent uponlocation, even days.

[0017] Millbrath et al [U.S. Pat. No. 5,401,634] disclose fluorochemicalemulsions comprised of a fluorochemical discontinuous phase and aqueouscontinuous phase with at least one specific binding species immobilizedon the droplets. The emulsions can include a “primer material” to couplespecific binding species to the fluorochemical droplets. The emulsionsmay be used in diagnostic procedures or biochemical reactors wherebinding of the immobilized specific binding species to its bindingpartner is desired. The droplets were envisioned to incorporate aspecies (e.g. dye) that is detectable by spectrophotometric,fluorometric or colormetric means. These inventors were focused upon invitro applications and were unconcerned with in vivo targeted drugdelivery. Moreover, they never conceived of the benefits of contactfacilitated drug or gene delivery achieved through ligand-targetedemulsion technology.

[0018] Magdassi et al, (published PCT application WO 95/03829) describedthe production and use of ligand-targeted oil emulsions in which drug is“dissolved, dispersed or solubilized inside the oil droplet, creating anovel drug targeting system”. The targeted particles provide an oilencapsulated depot of drug at the target site. Subsequent breakdown ofthe particles releases drug to the interstium. The agent is diluted inextracellular fluids and can migrate from the interstitium to the targetcell. Magdassi et al. do not conceive of the utility of contactfacilitated delivery of drug or genes from an outer surfactant layer totarget tissues. They do not recognize the importance or advantages of abiocompatible phospholipid surfactant layer amenable to exchangingconstituents with the target cell membrane. Contact facilitated drugdelivery with ligand-targeted emulsions places all of the drug into amonolayer surrounding the particle, ready to interact with the targetsurface. Mobility of the phospholipid monolayer over the particlesurface allows drug from all regions of the layer to migrate andinteract with target cell surfaces. Encapsulating drug within theparticle, as described by Magdassi et al. isolates the agent from theparticle surface and prevents contact facilitated delivery to the targetcell membrane.

[0019] Unger et al (U.S. Pat. No. 5,542,935) have described therapeuticdelivery systems for site-specific delivery of bioactive agents usinggas-filled perfluorocarbon microspheres. The microspheres contain atemperature activated gaseous precursor that becomes a gas uponactivation at a selected temperature. Once the microspheres have beenintroduced into the patient's body, a therapeutic compound may betargeted to specific tissues through the use of sonic energy, microwaveenergy, magnetic energy, or hyperthermia, which is directed to thetarget area and causes the microspheres to rupture and release thetherapeutic compound. Perfluorocarbons preferred includeperfluoromethane, perfluoroethane, perfluorobutane, perfluoropentane,perfluorohexane; even more preferably perfluoroethane, perfluoropentane,perfluoropropane, and perfluorobutane. Unger et al. notes that thelocalization of these particles for cavitation can be improved withconjugated ligand and the process of active targeting.

[0020] There remains a need for improved bioactive agent deliverysystems which provide enhanced efficiency of such bioactive agents totargeted tissues, cells or organs.

SUMMARY OF THE INVENTION

[0021] Among the several objects of the invention may be noted theprovision of novel compositions and methods for use in deliveringbioactive agents to targeted tissues or cells; the provision of suchcompositions which provide enhanced delivery of bioactive agents totargeted tissues or cells; the provision of such methods which providesenhanced intermingling and exchange of lipid components from one lipidsurface to the other thereby facilitating the exchange of bioactiveagents within or on the bioactive agent/emulsion surface to the targetcells or tissues; and the provision of such compositions and methodswhich may be readily practiced. Other objects will be in part apparentand in part pointed out hereinafter.

[0022] Briefly, in one aspect, the present invention is directed to acomposition for use in delivering a bioactive agent to targeted tissuesor cells comprising:

[0023] (a) site-specific targeting ligand;

[0024] (b) a lipid encapsulated oil in water emulsion; and

[0025] (c) a bioactive agent in or on the surface of the outer lipidmonolayer of said emulsion, said ligand being conjugated directly orindirectly to said emulsion and the composition providing facilitateddelivery of the bioactive agent through prolonged association andincreased contact of the ligand-bound, lipid encapsulated emulsionparticles with the lipid bilayer of said target tissues or cells. Theinvention is also directed to a method for improved delivery of abioactive agent to targeted tissues or cells comprising administeringthe above-noted compositions to said tissues or cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 is a graph showing that tissue factor-targeted doxorubicin(DXR) nanoparticles had greater anti-proliferative effects than freedoxorubicin and that this differential effect was greater at the lowerdosage of doxorubicin;

[0027]FIG. 2 is a graph showing the effectiveness of therapeuticpaclitaxel (Taxol) nanoparticle emulsions targeted to tissue factor onporcine aortic smooth muscle cells in accordance with the presentinvention; and

[0028]FIG. 3 is a graph showing the enhanced effectiveness oftherapeutic doxorubicin (DXR) or paclitaxel (Taxol) nanoparticleemulsions targeted to tissue factor on porcine aortic smooth musclecells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] In accordance with the present invention, it has now been foundthat improved compositions for use in delivering a bioactive agent totargeted tissues or cells may be formulated by combining (a) asite-specific targeting ligand; (b) a lipid encapsulated oil in wateremulsion; and (c) a bioactive agent in or on the surface of the outerlipid monolayer of the emulsion. The present invention thus relates toligand-targeted emulsions that incorporate biologically active agents onor in the outer monolayer of the lipid-based emulsion particle surface.These novel emulsions are particularly useful for the treatment ofdisease with biological agents that have improved risk/benefit profileswhen applied specifically to selected cells, tissues or organs.Site-directed, lipid encapsulated emulsions provide a unique opportunityto deliver potent bioactive agents, such as chemotherapeutic agents,nucleic acid-based therapy, protein-or peptide therapy and the like,with enhanced efficiency to targeted tissues through a unique form ofbioactive agent transfer into target cells, i.e. contact facilitateddelivery. Contact facilitated delivery of bioactive agents by targetedlipid encapsulated emulsions reflects the prolonged association andincreased contact of the ligand-bound, lipid-encapsulated particles withthe lipid bilayer of the target cell. Enhanced intermingling andexchange of lipid components from one lipid surface to the otherfacilitates the exchange of bioactive agents within or on thetherapeutic emulsion surface to the target cell. It will be understoodthat the bioactive agent is in or on the surface of the outer lipidmonolayer of the emulsion and is preferably, in accordance with theinvention, not carried or deposited in the interior of the emulsionparticles. It will also be understood that in accordance with thepresent invention, the emulsion must be nongaseous, i.e. should be aliquid emulsion.

[0030] Prolonged association of the emulsion particles with the surfaceof the targeted cell or tissue is in contradistinction to the transientinteraction that an unbound particle, existing free in extracellularbody fluids, would achieve. By binding the particle to the cell surface,the continued circulation of the nanoparticle through the body ishalted. The affixed particle is able to interact with the target cellsurface over an extended period of time. The exact amount of time may bevariable, but is meant to exceed that of more transient nontargetedcontact between particles and cell surfaces by orders of magnitude.

[0031] Targeted therapeutic emulsion may be delivered and concentratedat desired sites in vivo using active-targeting techniques. Activetargeting refers to ligand-directed, site-specific accumulation ofagents to cells, tissues or organs by localization and binding tomolecular epitopes, ie, receptors, lipids, peptides, cell adhesionmolecules, polysaccharides, biopolymers, and the like, presented on thesurface membranes of cells or within the extracellular matrix. A widevariety of ligands, including but not limited to antibodies, antibodyfragments, peptides, small molecules, polysaccharides, nucleic acids,aptamers, peptidomimetics, other mimetics and drugs alone or incombination may be utilized to specifically bind to cellular epitopesand receptors. These ligands may be attached covalently(direct-conjugation) or noncovalently (indirect conjugation) to theacoustic particle surface.

[0032] Avidin-biotin interactions are extremely useful, noncovalenttargeting systems that have been incorporated into many biological andanalytical systems and selected in vivo applications. Avidin has a highaffinity for biotin (10⁻¹⁵M) facilitating rapid and stable binding underphysiological conditions. Targeted systems utilizing this approach areadministered in two or three steps, depending on the formulation.Typically, a biotinylated ligand, such as a monoclonal antibody, isadministered first and “pretargeted” to the unique molecular epitopes.Next, avidin is administered, which binds to the biotin moiety of the“pretargeted” ligand. Finally, the biotinylated agent is added and bindsto the unoccupied biotin-binding sites remaining on the avidin therebycompleting the ligand-avidin-emulsion “sandwich”. The avidin-biotinapproach can avoid accelerated, premature clearance of targeted agentsby the MPS system secondary to the presence of surface antibody.Additionally, avidin, with four, independent biotin binding sitesprovides signal amplification and improves detection sensitivity.

[0033] Targeting ligands may be chemically attached to the surface ofacoustic or emulsion particles by a variety of methods depending uponthe nature of the ligand and composition of the particle surface. Aligand is a molecule that binds to another molecule, and as used in thisapplication refers to a small targeting molecule that binds specificallyto another molecule on a biological surface separate and distinct fromthe emulsion particle itself. The reaction does not require nor excludea molecule that donates or accepts a pair of electrons to form acoordinate covalent bond with a metal atom of a coordination complex.Conjugations may be performed before or after the emulsion particle iscreated depending upon the ligand employed. Direct chemical conjugationof ligands to proteinaceous agents often take advantage of numerousamino-groups (e.g. lysine) inherently present within the surface.Alternatively, functionally active chemical groups such aspyridyldithiopropionate, maleimide or aldehyde may be incorporated intothe surface as chemical “hooks” for ligand conjugation after theparticles are formed. Another common post-processing approach is toactivate surface carboxylates with carbodiimide prior to ligandaddition. The selected covalent linking strategy is primarily determinedby the chemical nature of the ligand. Monoclonal antibodies and otherlarge proteins may denature under harsh processing conditions; whereas,the bioactivity of carbohydrates, short peptides, aptamers, drugs orpeptidomimetics often can be preserved. To ensure high ligand bindingintegrity and maximize targeted particle avidity flexible polymer spacerarms, e.g. polyethylene glycol, amino acids or simple caproate bridges,can be inserted between an activated surface functional group and thetargeting ligand. These extensions can be 10 nm or longer and minimizeinterference of ligand binding by particle surface interactions.

[0034] Monoclonal antibody and fragments: Rapid expansion of themonoclonal antibody industry has prepared the stage for the clinicalsuccess of site-targeted agents by providing a plethora of ligands thatcan be directed against a wide spectrum of pathologic molecularepitopes. Antibodies or their fragments may be from several classesincluding IgG, IgM, IgA, IgE or IgD. Immunoglobin-γ (IgG) classmonoclonal antibodies have been most often conjugated to liposomes,emulsions and other microbubble particles to provide active,site-specific targeting. These proteins are symmetric glycoproteins (MWca. 150,000 daltons) composed of identical pairs of heavy and lightchains. Hypervariable regions at the end of each of two arms provideidentical antigen-binding domains. A variably sized branchedcarbohydrate domain is attached to complement-activating regions, andthe hinge area contains particularly accessible interchain disulfidebonds that may be reduced to produce smaller fragments.

[0035] Bivalent F(ab′)₂ and monovalent F(ab) fragments are derived fromselective cleavage of the whole antibody by pepsin or papain digestion,respectively. Elimination of the Fc region greatly diminishes theimmunogenicity of the molecule, diminishes nonspecific liver uptakesecondary to bound carbohydrate, and reduces complement activation andresultant antibody-dependent cellular toxicity. Complement fixation andassociated cellular cytotoxicity can be detrimental when the targetedsite must be preserved or beneficial when recruitment of host killercells and target-cell destruction is desired (e.g. anti-tumor agents).

[0036] Most monoclonal antibodies are of murine origin and areinherently immunogenic to varying extents in other species. Humanizationof murine antibodies through genetic engineering or other combinatorialchemical methods have led to development of chimeric ligands withimproved biocompatibility and longer circulatory half-lives. The bindingaffinity of recombinant antibodies to targeted molecular epitopes can beoccasionally improved with selective site-directed mutagenesis of thebinding idiotype.

[0037] Phage display: Phage display techniques are now used to producerecombinant human monoclonal antibody fragments against a large range ofdifferent antigens without involving antibody-producing animals. Ingeneral, cloning creates large genetic libraries of corresponding DNA(cDNA) chains deducted and synthesized by means of the enzyme “reversetranscriptase” from total messenger RNA (mRNA) of human B lymphocytes.Immunoglobulin cDNA chains are amplified by PCR (polymerase chainreaction) and light and heavy chains specific for a given antigen areintroduced into a phagemid vector. Transfection of this phagemid vectorinto the appropriate bacteria results in the expression of a scFvimmunoglobulin molecule on the surface of the bacteriophage.Bacteriophages expressing specific immunoglobulin are selected byrepeated immunoadsorption/phage multiplication cycles against desiredantigens (e.g., proteins, peptides, nuclear acids, and sugars).Bacteriophages strictly specific to the target antigen are introducedinto an appropriate vector, (e.g., Escherichia coli, yeast, cells) andamplified by fermentation to produce large amounts of human antibodyfragments with structures very similar to natural antibodies. Althoughthis technology is still in early stages of development, it has alreadypermitted the production of unique ligands for targeting and therapeuticapplications. (de Bruin et al., Selection of high-affinity phageantibodies from pahage display libraries. Nat Biotechnol. 1999;17:397-399; Stadler, Antibody production without animals. Dev BiolStand. 1999; 101:45-48; Wittrup, Phage on display, Trends Biotechnol.1999; 17:423-424; Sche et al., Display cloning: functionalidentification of natural product receptors using cDNA-phage display.Chem Biol. 1999; 6:(707-716).

[0038] Peptides: Peptides, like antibodies, may have high specificityand epitope affinity for use as vector molecules for targeted contrastagents. These may be small peptides (5 to 10 amino acids) specific for aunique receptor sequences (e.g. such as the RGD epitope of the plateletGIIbIIIa receptor) (Wright et al., Evaluation of New thrombus-specificultrasound contrast agents. Acad Radiol. 1998; 5 (Supp 1): S240-S242) orlarger, biologically active hormones such as cholecystokinin (Reimer etal., Pancreatic receptors: initial feasibility studies with a targetedcontrast agent for MR imaging; Radiology, 1994; 193:527-531). Smallerpeptides potentially have less inherent immunogenicity than nonhumanizedmurine antibodies. Peptides or peptide (nonpeptide) analogues of celladhesion molecules, cytokines, selectins, cadhedrins, Ig superfamily,integrins and the like may be utilized for targeted therapeuticdelivery.

[0039] Asialoglycoproteins and polysaccharides: Asialoglycoproteins(ASG) have been used for liver-specific applications due to their highaffinity for ASG receptors located uniquely on hepatocytes (Reimer etal., Preclinical assessment of hepatocyte-targeted MR constrast agentsin stable human liver cell cultures, J Magn Reson Imaging, 1998;8:687-698 and Leveille-Webster et al., Use of an asialoglycoproteinreceptor-targeted magnetic resonance agent to study changes in receptorbiology during liver regeneration and endotoxemia in rats., Hepatology1996; 23:1631-1641). ASG directed agents (primarily MR agents conjugatedto iron oxides) have been used to detect primary and secondary hepatictumors as well as benign, diffuse liver disease such as hepatitis(Reimer et al., Dynamic signal intensity changes in liver withsuperparamagnetic MR constrast agents., J Magn Reson Imaging, 1992;2:177-181 and Reimer et al., Receptor-directed contrast agents for MRimaging preclinical evaluation with affinity assays., Radiology, 1992;182:565-569). The ASG receptor is highly abundant on hepatocytes,approximately 500,000 per cell, rapidly internalizes and is subsequentlyrecycled to the cell surface. Polysaccharides such as arabinogalactanmay also be utilized to localize agents to hepatic targets.Arabinogalactan has multiple terminal arabinose groups that display highaffinity for ASG hepatic receptors (Leveille-Webster et al., supra andSmall et al., Enhancement effects of a hepatocyte receptor-specific MRcontrast agent in an animal model., J Magn Reson Imaging, 1994;4:325-330).

[0040] Aptamers: Aptamers are high affinity, high specificity RNA orDNA-based ligands produced by in vitro selection experiments (SELEX:systematic evolution of ligands by exponential enrichment) (Small etal., supra).

[0041] Aptamers are generated from random sequences of 20 to 30nucleotides, selectively screened by absorption to molecular antigens orcells, and enriched to purify specific high affinity binding ligands. Toenhance in vivo stability and utility, aptamers are generally chemicallymodified to impair nuclease digestion and to facilitate conjugation withdrugs, labels or particles. Other, simpler chemical bridges oftensubstitute nucleic acids not specifically involved in the ligandinteraction. In solution aptamers are unstructured but can fold andenwrap target epitopes providing specific recognition. The uniquefolding of the nucleic acids around the epitope affords discriminatoryintermolecular contacts through hydrogen bonding, electrostaticinteraction, stacking, and shape complementarity. In comparison withprotein-based ligands, aptamers are stable, are more conducive to heatsterilization, and have lower immunogenicity. Aptamers are currentlyused to target a number of clinically relevant pathologies includingangiogenesis, activated platelets, and solid tumors and their use isincreasing. The clinical effectiveness of aptamers as targeting ligandsfor therapeutic emulsion particles may be dependent upon the impact ofthe negative surface charge imparted by nucleic acid phosphate groups onclearance rates. Previous research with lipid-based particles suggestthat negative zeta potentials markedly decrease liposome circulatoryhalf-life, whereas, neutral or cationic particles have similar, longersystemic persistence.

[0042] The oil phase of the oil in water emulsion may be a vegetableoil, medium chain triglycerides (MCT) or any other oil, with increasedor decreased polarity and hydrophobicity or fluorochemical liquid.Suitable fluorochemical liquids include straight and branched chain andcyclic perfluorocarbons, straight and branched chain and cyclicperfluoro tertiary amines, straight and branched chain and cyclicperfluoro ethers and thioethers, halofluorocarbons and polymericperfluoro ethers and the like. Although up to 50% hydrogen-substitutedcompounds can be used, perhalo compounds are preferred. Most preferredare perfluorinated compounds.

[0043] Although any fluorochemical liquid i.e. a substance which is aliquid at about 20 degree.C at atmospheric pressure, can be used toprepare a fluorochemical emulsion of the present invention, for manypurposes emulsions with longer extended stability are preferred. Inorder to obtain such emulsions, fluorochemical liquids with boilingpoints above 30.degree. C are preferred. Preferably the fluorochemicalliquids have boiling points above 50 C., and most preferred arefluorochemical liquids with boiling points above about 90 C.

[0044] Useful perfluorocarbon emulsions are disclosed in U.S. Pat. Nos.4,927,623, 5,077,036, 5,114,703, 5,171,755, 5,304,325, 5,350,571,5,393,524, and 5,403,575 and include those in which the perfluorocarboncompound is perfluorodecalin, perfluorooctane, perfluorodichlorooctane,perfluoro-n-octyl bromide, perfluoroheptane, perfluorodecane,perfluorocyclohexane, perfluoromorpholine, perfluorotripropylamine,perfluortributylamine, perfluorodimethylcyclohexane,perfluorotrimethylcyclohexane, perfluorodicyclohexyl ether,perfluoro-n-butyltetrahydrofuran, and compounds that are structurallysimilar to these compounds and are partially or fully halogenated(including at least some fluorine substituents) or partially or fullyperfluorinated including perfluoroalkylated ether, polyether or crownether.

[0045] Emulsifying agents, for example surfactants, may be used tofacilitate the formation of emulsions. Typically, aqueous phasesurfactants have been used to facilitate the formation of emulsions offluorochemical liquids. A variety of lipid surfactants may beincorporated into the lipid monolayer preferably natural or syntheticphospholipids, but also fatty acids, cholesterols, lysolipids,sphingomyelins, tocopherols, glucolipids, stearylamines, cardiolipins, alipid with ether or ester linked fatty acids, polymerized lipids, andlipid conjugated polyethylene glycol. The preferred surfactants for usein the practice of the invention are phospholipids and cholesterol.Other known surfactants such as Pluronic F-68, Hamposyl.™ L30 (W.R.Grace Co., Nashua, N.H.), sodium dodecyl sulfate, Aerosol 413 (AmericanCyanamid Co., Wayne, N.J.), Aerosol 200 (American Cyanamid Co.),Lipoproteol.™ LCO (Rhodia Inc., Mammoth, N.J.), Standapol.™ SH 135(Henkel Corp., Teaneck, N.J.), Fizul.™ 10-127 (Finetex Inc., ElmwoodPark, N.J.), and Cyclopol.™ SBFA 30 (Cyclo Chemicals Corp., Miami,Fla.); amphoterics, such as those sold with the trade names: Deriphat.™170 (Henkel Corp.), Lonzaine.™ JS (Lonza, Inc.), Niranol.™ C2N-SF(Miranol Chemical Co., Inc., Dayton, N.J.), Amphoterge.™ W2 (Lonza,Inc.), and Amphoterge.™ 2WAS (Lonza, Inc.); non-ionics, such as thosesold with the trade names: Pluronic.™ F-68 (BASF Wyandotte, Wyandotte,Mich.), Pluronic.™ F-127 (BASF Wyandotte), Brij.™ 35 (ICI Americas;Wilmington, Del.), Triton.™ X-100 (Rohm and Haas Co., Philadelphia,Pa.), Brij.™ 52 (ICI Americas), Span.™ 20 (ICI Americas), Generol.™ 122ES (Henkel Corp.), Triton.™ N-42 (Rohm and Haas Co.,), Triton.™ N-101(Rohm and Haas Co.,), Triton.™ X-405 (Rohm and tlaas Co.,), Tween.™ 80(ICI Americas), Tween.™ 85 (ICI Americas), and Brij.™ 56 (ICI Americas)and the like may be used. These surfactants' are used alone or incombination in amounts of 0.10 to 5.0% by weight to assist instabilizing the emulsions.

[0046] Fluorinated surfactants which are soluble in the fluorochemicalliquid to be emulsified can also be used. Suitable fluorochemicalsurfactants include perfluorinated alkanoic acids such asperfluorohexanoic and perfluorooctanoic acids and amidoaminederivatives. These surfactants are generally used in amounts of 0.01 to5.0% by weight, and preferably in amounts of 0.1 to 1.0%. Other suitablefluorochemical surfactants include perfluorinated alcohol phosphateesters and their salts; perfluorinated sulfonamide alcohol phosphateesters and their salts; perfluorinated alkyl sulfonamide alkylenequaternary ammonium salts; N,N-(carboxyl-substituted lower alkyl)perfluorinated alkyl sulfonamides; and mixtures thereof. As used herein,the term “perfluorinated” means that the surfactant contains at leastone perfluorinated alkyl group.

[0047] Suitable perfluorinated alcohol phosphate esters include the freeacids of the diethanolamine salts of mono- andbis(1H,1H,2H,2H-perfluoroalkyl)phosphates. The phosphate salts,available under the tradename “Zonyl RP” (E.I. Dupont de Nemours andCo., Wilmington, Del.), are converted to the corresponding free acids byknown methods. Suitable perfluorinated sulfonamide alcohol phosphateesters are described in U.S. Pat. No. 3,094,547. Suitable perfluorinatedsulfonamide alcohol phosphate esters and salts of these includeperfluoro-n-octyl-N-ethylsulfonamidoethyl phosphate,bis(perfluoro-n-octyl-N-ethylsulfonamidoethyl)phosphate, the ammoniumsalt of bis(perfluoro-n-octyl-N-ethylsulfonamidoethyl)phosphate,bis(perfluorodecyl-N-ethylsulfonamidoethyl)-phosphate andbis(perfluorohexyl-N ethylsulfonamidoethyl)-phosphate. The preferredformulations use phosphatidylcholine,derivatized-phosphatidylethanolamine and cholesterol as the aqueoussurfactant.

[0048] Lipid encapsulated emulsions can be formulated with cationiclipids in the surfactant layer that facilitate the adhesion of nucleicacid material to particle surfaces. Cationic lipids may include but arenot limited to DOTMA,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride; DOTAP,1,2-dioleoyloxy-3-(trimethylammonio)propane; DOTB,1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl-sn-glycerol,1,2-diacyl-3-trimethylammonium-propane;1,2-diacyl-3-dimethylammonium-propane; 1,2-diacyl-sn-glycerol-3-ethylphosphocholine; and3β-[N′,N′-dimethylaminoethane)-carbamol]cholesterol-HCl may be used. Ingeneral the molar ratio of cationic lipid to non-cationic lipid in thelipid surfactant monolayer may be, for example, 1:1000 to 2:1,preferably, between 2:1 to 1:10, more preferably in the range between1:1 to 1:2.5 and most preferably 1:1 (ratio of mole amount cationiclipid to mole amount non-cationic lipid, e.g., DPPC). A wide variety oflipids may comprise the non-cationic lipid component of the emulsionsurfactant, particularly dipalmitoylphosphatidylcholine,dipalmitoylphosphatidylethanolamine,1,2-diacyl-sn-glycerol-3-phosphoethanolamine, cholesterol ordioleoylphosphatidylethanolamine in addition to those previouslydescribed. In lieu of cationic lipids as described above, lipids bearingcationic polymers such as polylysine or polyarginine may also beincluded in the lipid surfactant and afford binding of a negativelycharged therapeutic, such as genetic material or analogues there of, tothe outside of the emulsion particles.

[0049] Release of nucleic acid constructs from cationic lipid emulsionscan be induced by the incorporation of anionic lipids from adjacentlipid monolayers and faciliated by the prolonged contact andinteractions afforded by ligand-targeted emulsions. A similar phenomenahas been reported when nucleic acid linked cationic liposomes becomeintracellular. These anionic lipids form a charged ion-pair with thecationic lipids leading to the displacement of oligonucleotide into thesurrounding medium. Delivery of genetic material adsorbed onto thesurface of cationic emulsions can be enhanced by ligand-targetedemulsions and contact facilitated delivery. Increased interaction andexchange of lipids from the target cell surface to the outer membrane ofthe therapeutic particle will facilitate the transfer and release ofoligonucleotide.

[0050] In accordance with the present invention, the site-specifictargeting ligand may be directly or indirectly conjugated to the surfaceof the emulsion particles. Direct conjugation of ligand to the emulsionparticles refers to the preparation of a ligand-particle complex beforeadministration to a patient wherein the ligand is either adsorbedthrough ionic, electrostatic, hydrophobic or other noncovalent means tothe particle surface (e.g. acylated-antibody), or chemically linked tothe surface through covalent bonds to a component of the lipid surfacesuch as a “primer material” (e.g. thio-ether or ester bond), orintrinsically incorporated into the lipid surfactant membrane as acomponent of the membrane (e.g. a lipid derivatized to a peptidomimeticagent).

[0051] Indirect conjugation refers to the use of avidin biotin whereinthe complex is formed in vivo in two or more steps. For example, byadministering the biotinylated antibody first, followed by avidin, andfollowed by the biotinylated emulsion particle. Any other sequentialmultistep chemical linking system that could be used in vivo isenvisioned to produce the same end result, i.e. the close and specificapposition of the emulsion particle to a targeted cell or tissuesurface.

[0052] The emulsions of the present invention may be prepared by varioustechniques. One method is sonication of a mixture of oil orfluorochemical liquid and an aqueous solution containing a suitable“primer” material and/or specific binding species. Generally, thesemixtures include a surfactant. Cooling the mixture being emulsified,minimizing the concentration of surfactant, and buffering with a salinebuffer will typically maximize both retention of specific bindingproperties and the coupling capacity of the primer material. Thesetechniques provide excellent emulsions with high activity per unit ofabsorbed primer material or specific binding species.

[0053] When high concentrations of a primer material or specific bindingspecies coated on lipid emulsions, the mixture should be heated duringsonication and have a relatively low ionic strength and moderate to lowpH. Too low an ionic strength, too low a pH or too much heat may causesome degradation or loss of all of the useful binding properties of thespecific binding species or the coupling capacity of the “primer”material. Careful control and variation of the emulsification conditionscan optimize the properties of the “primer” material or the specificbinding species while obtaining high concentrations of coating.

[0054] Carbohydrate-bearing lipids may be employed for in vivotargeting, as described in U.S. Pat. No. 4,310,505, the disclosures ofwhich are hereby incorporated herein by reference, in their entirety.

[0055] While preparation of emulsions by sonication has been acceptable,some degree of variability in particle size distribution may beobserved. An alternative method of making the emulsions involvesdirecting high pressure streams of mixtures containing the aqueoussolution, a “primer” material or the specific binding species, the oilor fluorocarbon liquid and a surfactant (if any) so that they impact oneanother to produce emulsions of narrow particle size and distribution.The Microfluidizer™ apparatus (Microfluidics, Newton, Mass.) can be usedto make the preferred emulsions. The apparatus is also useful topost-process emulsions made by sonication or other conventional methods.Feeding a stream of emulsion droplets through the Microfluidizer™apparatus yields formulations of small size and narrow particle sizedistribution.

[0056] Emulsifying and/or solubilizing agents may also be used inconjunction with emulsions. Such agents include, but are not limited to,acacia, cholesterol, diethanolamine, glyceryl monostearate, lanolinalcohols, lecithin, mono- and di-glycerides, mono-ethanolamine, oleicacid, oleyl alcohol, poloxamer, peanut oil, palmitic acid,polyoxyethylene 50 stearate, polyoxyl 35 castor oil, polyoxyl 10 oleylether, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbate20, polysorbate 40, polysorbate 60, polysorbate 80, propylene glycoldiacetate, propylene glycol monostearate, sodium lauryl sulfate, sodiumstearate, sorbitan mono-laurate, sorbitan mono-oleate, sorbitanmono-palmitate, sorbitan monostearate, stearic acid, trolamine, andemulsifying wax. All lipids with perfluoro fatty acids as a component ofthe lipid in lieu of the saturated or unsaturated hydrocarbon fattyacids found in lipids of plant or animal origin may be used. Suspendingand/or viscosity-increasing agents that may be used with emulsionsinclude, but are not limited to, acacia, agar, alginic acid, aluminummono-stearate, bentonite, magma, carbomer 934P, carboxymethylcellulose,calcium and sodium and sodium 12, carrageenan, cellulose, dextrin,gelatin, guar gum, hydroxyethyl cellulose, hydroxypropylmethylcellulose, magnesium aluminum silicate, methylcellulose, pectin,polyethylene oxide, polyvinyl alcohol, povidone, propylene glycolalginate, silicon dioxide, sodium alginate, tragacanth, and xanthum gum.

[0057] Useful emulsions may have a wide range of nominal particlediameters, e.g., from as small as 0.01 microns to as large as 10microns, preferably 0.1 to 0.5 micron diameter. The emulsion particlesizes can be controlled and varied by modifications of theemulsification techniques and the chemical components.

[0058] The specific binding species (i.e. targeting ligand) may beimmobilized on the encapsulating lipid monolayer by direct adsorption tothe oil/aqueous interface or using a “primer material”. A “primermaterial” is any surfactant compatible compound incorporated in theparticle to chemically couple with or adsorb a specific binding ortargeting species i.e. any constituent or derivatized constituentincorporated into the emulsion lipid surfactant layer that could bechemically utilized to form a covalent bond between the particle andtargeting ligand or a component of the targeting ligand (if it hassubunits). The preferred result is achieved by forming an emulsion withan aqueous continuous phase and a biologically active ligand adsorbed orconjugated to the “primer material” at the interface of the continuousand discontinuous phases. Naturally occurring or synthetic polymers withamine, carboxyl, mercapto, or other functional groups capable ofspecific reaction with coupling agents and highly charged polymers maybe utilized in the coupling process. The specific binding species (e.g.antibody) may be immobilized on the emulsion particle surface by directadsorption or by chemical coupling. Examples of specific binding specieswhich can be immobilized by direct adsorption include small peptides,peptidomimetics, or polysaccharide-based agents. To make such anemulsion the specific binding species may be suspended or dissolved inthe aqueous phase prior to formation of the emulsion. Alternatively, thespecific binding species may be added after formation of the emulsionand incubated with gentle agitation at room temperature (25° C.) in a pH7.0 buffer (typically phosphate buffered saline) for 1.2 to 18 hours.

[0059] Where the specific binding species is to be coupled to a “primermaterial”, conventional coupling techniques may be used. The specificbinding species may be covalently bonded to “primer material” withcoupling agents using methods which are known in the art. One type ofcoupling agent uses a carbodiimide such as 1-ethyl-3-(3-N,Ndimethylaminopropyl)carbodiimide hydrochloride or1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemethyl-p-toluenesulfonate. The priimer material may bephosphatidylethanolamine, N-caproylamine phosphatidylethanolamine,N-dodecanylamine phosphatidylethanolamine, phosphotidylthioethanol,1,2-diacyl-sn-glycerol-3-phosphoethanolamine-N-[4-p-maleimidephenyl)-butyramide,N-succinyl-phosphatidylethanolamine,N-glutaryl-phosphatidylethanolamine,N-dodecanyl-phosphatidylethanolamine,N-biotinyl-phosphatidylethanolamine,N-biotinylcaproyl-phosphatidylethanolamine, and phosphatidylethyleneglycol. Other suitable coupling agents include aldehyde coupling agentshaving either ethylenic unsaturation such as acrolein, methacrolein, or2-butenal, or having a plurality of aldehyde groups such asglutaraldehyde, propanedial or butanedial. Other coupling agents include2-iminothiolane hydrochloride, bifunctional N-hydroxysuccinimide esterssuch as disuccinimidyl subsrate, disuccinimidyl tartrate,bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, disuccinimidylpropionate, ethylene glycolbis(succinimidyl succinate);heterobifunctional reagents such asN-(5-azido-2-nitrobenzoyloxy)succinimide, p-azidophenylbromide,p-azidophenylglyoxal, 4-fluoro-3-nitrophenylazide,N-hydroxysuccinimidyl-4-azidobenzoate, m-maleimidobenzoylN-hydroxysuccinimide ester, methyl-4-azidophenylglyoxal,4-fluoro-3-nitrophenyl azide, N-hydroxysuccinimidyl-4-azidobenzoatehydrochloride, p-nitrophenyl 2-diazo-3,3,3-trifluoropropionate,N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate, succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, succinimidyl4-(p-maleimidophenyl)butyrate,N-succinimidyl(4-azidophenyldithio)propionate, N-succinimidyl3-(2-pyridyldithio)propionate, N-(4-azidophenylthio)phthalamide;homobifunctional reagents such as 1,5-difluoro-2,4-dinitrobenzene,4,4′-difluoro-3,3′-dinitrodiphenylsulfone,4,4′-diisothiocyano-2,2′-disulfonic acid stilbene,p-phenylenediisothiocyanate, carbonylbis(L-methionine p-nitrophenylester), 4,4′-dithiobisphenylazide, erythritolbiscarbonate andbifunctional imidoesters such as dimethyl adipimidate hydrochloride,dimethyl suberimidate, dimethyl 3,3′-dithiobispropionimidatehydrochloride and the like. Covalent bonding of a specific bindingspecies to the “primer material” can be carried out with the abovereagents by conventional, well-known reactions, for example, in theaqueous solutions at a neutral pH, at temperatures of less than 25° C.for 1 hour to overnight.

[0060] Targeted therapeutic emulsions may incorporate bioactive agents(e.g drugs, genetic materials, radioactive isotopes, or combinationsthereof) in their native form or derivatized with hydrophobic or chargedmoieties to enhance incorporation or adsorption to the ligand targetedparticle. Such therapeutics may include, but are not limited toantineoplastic agents, such as platinum compounds (e.g., spiroplatin,cisplatin, and carboplatin), methotrexate, fluorouracil, adriamycin,mitomycin, ansamitocin, bleomycin, cytosine arabinoside, arabinosyladenine, mercaptopolylysine, vincristine, busulfan, chlorambucil,melphalan (e.g., PAM, L-PAM or phenylalanine mustard), mercaptopurine,mitotane, procarbazine hydrochloride dactinomycin (actinomycin D),daunorubicin hydrochloride, doxorubicin hydrochloride, paclitaxel andother taxenes, rapamycin, manumycin A, TNP-470, plicamycin(mithramycin), aminoglutethimide, estramustine phosphate sodium,flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate,testolactone, trilostane, amsacrine (m-AMSA), asparaginase(L-asparaginase) Erwina asparaginase, interferon .alpha.-2a, interferonalpha.-2b, teniposide (VM-26), vinblastine sulfate (VLB), vincristinesulfate, bleomycin sulfate, hydroxyurea, procarbazine, and dacarbazine;mitotic inhibitors such as etoposide, colchicine, and the vincaalkaloids, radiopharmaceuticals such as radioactive iodine andphosphorus products; hormones such as androgens, progestins, estrogensand antiestrogens; antihelmintics, antimalarials, and antituberculosisdrugs; biologicals such as immune serums, antitoxins and antivenoms;rabies prophylaxis products; bacterial vaccines; viral vaccines;aminoglycosides; respiratory products such as xanthine derivativestheophylline and aminophylline; thyroid agents such as iodine productsand anti-thyroid agents; cardiovascular products including chelatingagents and mercurial diuretics and cardiac glycosides; glucagon; bloodproducts such as parenteral iron, hemin, hematoporphyrins and theirderivatives; biological response modifiers such as muramyldipeptide,muramyltripeptide, microbial cell wall components, lymphokines (e.g.,bacterial endotoxin such as lipopolysaccharide, macrophage activationfactor), sub-units of bacteria (such as Mycobacteria, Corynebacteria),the synthetic dipeptide N-acetyl-muramyl-L-alanyl-D-isoglutamine;anti-fungal agents such as ketoconazole, nystatin, griseofulvin,flucytosine (5-fc), miconazole, amphotericin B, ricin, cyclosporins, and.beta.-lactam antibiotics (e.g., sulfazecin); hormones such as growthhormone, melanocyte stimulating hormone, estradiol, beclomethasonedipropionate, betamethasone, betamethasone acetate and betamethasonesodium phosphate, vetamethasone disodium phosphate, vetamethasone sodiumphosphate, cortisone acetate, dexamethasone, dexamethasone acetate,dexamethasone sodium phosphate, flunisolide, hydrocortisone,hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodiumphosphate, hydrocortisone sodium succinate, methylprednisolone,methylprednisolone acetate, methylprednisolone sodium succinate,paramethasone acetate, prednisolone, prednisolone acetate, prednisolonesodium phosphate, prednisolone tebutate, prednisone, triamcinolone,triamcinolone acetonide, triamcinolone diacetate, triamcinolonehexacetonide, fludrocortisone acetate, oxytocin, vassopressin, and theirderivatives; vitamins such as cyanocobalamin neinoic acid, retinoids andderivatives such as retinol palmitate, and alpha.-tocopherol; peptides,such as manganese super oxide dismutase; enzymes such as alkalinephosphatase; anti-allergic agents such as amelexanox; anti-coagulationagents such as phenprocoumon and heparin; circulatory drugs such aspropranolol; metabolic potentiators such as glutathione; antitubercularssuch as para-aminosalicylic acid, isoniazid, capreomycin sulfatecycloserine, ethambutol hydrochloride ethionamide, pyrazinamide,rifampin, and streptomycin sulfate; antivirals such as acyclovir,amantadine azidothymidine (AZT, DDI, Foscarnet, or Zidovudine),ribavirin and vidarabine monohydrate (adenine arabinoside, ara-A);antianginals such as diltiazem, nifedipine, verapamil, erythritoltetranitrate, isosorbide dinitrate, nitroglycerin (glyceryl trinitrate)and pentaerythritol tetranitrate; anticoagulants such as phenprocoumon,heparin; antibiotics such as dapsone, chloramphenicol, neomycin,cefaclor, cefadroxil, cephalexin, cephradine erythromycin, clindamycin,lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin,dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin,nafcillin, oxacillin, penicillin including penicillin G and penicillinV, ticarcillin rifampin and tetracycline; antiinflammatories such asdiflunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid,naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac,tolmetin, aspirin and salicylates; antiprotozoans such as chloroquine,hydroxychloroquine, metronidazole, quinine and meglumine antimonate;antirheumatics such as penicillamine; narcotics such as paregoric;opiates such as codeine, heroin, methadone, morphine and opium; cardiacglycosides such as deslanoside, digitoxin, digoxin, digitalin anddigitalis; neuromuscular blockers such as atracurium mesylate, gallaminetriethiodide, hexafluorenium bromide, metocurine iodide, pancuroniumbromide, succinylcholine chloride (suxamethonium chloride), tubocurarinechloride and vecuronium bromide; sedatives (hypnotics) such asamobarbital, amobarbital sodium, aprobarbital, butabarbital sodium,chloral hydrate, ethchlorvynol, ethinamate, flurazepam hydrochloride,glutethimide, methotrimeprazine hydrochloride, methyprylon, midazolamhydrochloride, paraldehyde, pentobarbital, pentobarbital sodium,phenobarbital sodium, secobarbital sodium, talbutal, temazepam andtriazolam; local anesthetics such as bupivacaine hydrochloride,chloroprocaine hydrochloride, etidocaine hydrochloride, lidocainehydrochloride, mepivacaine hydrochloride, procaine hydrochloride andtetracaine hydrochloride; general anesthetics such as droperidol,etomidate, fentanyl citrate with droperidol, ketamine hydrochloride,methohexital sodium and thiopental sodium; and radioactive particles orions such as strontium, iodide rhenium and yttrium.

[0061] In addition, the ligand itself, such as an antibody, peptidefragment, or a mimetic of a biologically active ligand may contribute tothe inherent therapeutic effects, either as an antagonistic oragonistic, when bound to specific epitopes. As an example, antibodyagainst ανβ3 integrin on neovascular endothelial cells has been shown totransiently inhibit growth and metastasis of solid tumors. The efficacyof therapeutic emulsion particles directed to the ανβ3 integrin mayresult from the improved antagonistic action of the targeting ligand inaddition to the effect of the therapeutic agents incorporated anddelivered by particle itself Thus, in another embodiment of theinvention, the targeting ligand and bioactive agent may be constitutedby a single component which functions both to target the ligand and toprovide the bioactive agent to the desired site. As in the otherembodiments previously described, when the targeting ligand andbioactive agent are constituted by a single component, such componentmust still be on or in the surface of the outer monolayer of the lipidencapsulated oil in water emulsion.

[0062] The following non-limiting examples illustrate the practice ofthe invention and the use of the lipid encapsulated oil in wateremulsions with bioactive agents which are difficult to administer. Byway of background for the examples set forth below, many potentpharmaceutical agents are difficult to administer systemically topatients in a safe and efficacious manner either because of secondarycomplications due to toxicity of the parent drug or the formulationexcipients. Many of these drugs are inherently hydrophobic, such aspaclitaxel and analogues thereof. Other compounds are water soluble withlipophilic regions in the parent drug (e.g. doxorubicin) or are amenableto the addition of lipophilic moieties. In this disclosure, we revealthe enhanced effectiveness of such drugs incorporated into or on thesurfactant surface of emulsions, such as perfluorocarbon emulsions,targeted specifically to tissues, organs or cells through contactfacilitated delivery.

[0063] Doxorubicin, is a antineoplastic agent that is incorporated intothe treatment regimens of a wide variety of human tumors. Daunorubicinand its 14-hydroxy derivative, doxorubicin, are anthracyclineantibiotics produced by the fungus streptomyces peucetius. Doxorubicinis highly water soluble and structurally consists of an aglycon,adriamycinone, combined with an aminosugar, daunosamine Doxorubicindamages DNA by intercalation of the anthracycline portion, metal ionchelation, or by generation of free radicals. Doxorubicin has also beenshown to inhibit DNA topoisomerase II which is critical to DNA function.Cytotoxic activity is cell cycle phase-nonspecific. The therapeuticactivity of doxorubicin is well validated, and equally well known areits adverse effects, including hair loss, mouth sores, nausea, vomiting,lowered blood counts (WBCs, RBCs and platelets), cardiotoxicity and skindamage secondary to extravasation during intravenous infusion. Theanthracycline aspect of the molecule promotes the interaction ofdoxorubicin with acyl regions of lipid monolayers and bilayers,moderately with neutral layers and strongly with positive or negativelycharged membranes hydrophobic and electrostatic interactions.Doxorubicin has been encapsulated into liposomes with demonstrableimprovements in organ distribution and effectiveness of the drug as wellas a decrease of its cardiotoxicity versus free doxorubicin. The overallbenefits of a variety of liposomal doxorubicin versus free drugindependent of vesicle composition or formulation process are todecrease systemic toxicity symptoms associated with high peakconcentrations of drug and to prolong the pharmacological half-life ofthe compound.

[0064] In addition to liposomal Doxil, doxorubicin and its conjournershave been incorporated into a variety of emulsions and polymericconstructs with some success. Like the liposomal formulations, theseformulations strive to administer high payloads of drug with diminishedpeak concentrations and prolonged systemic half-life. For example,doxorubicin has been prepared as micelles composed of poly(ethyleneglycol)-poly(beta-benzyl-L-aspartate) block copolymer (PEG-PBLA) by ano/w emulsion method with a substantial drug loading level (15 to 20 w/w%). Doxorubicin was chemically conjugated to a terminal end group ofpoly(D,L-lactic-co-glycolic acid) [PLGA] and the doxorubicin-PLGAconjugate was formulated into nanoparticles to sustain the release ofdoxorubicin. Using sonication and a detergent, iodinated poppy seed oil(IPSO) has been mixed with aqueous solution epirubicin to yield awater-in-oil emulsion that is passed through a microporous glassmembrane into saline water-in-oil-in-water emulsion (W/O/W) thatconsists of IPSO microdroplets. A doxorubicin suspension has beenprepared by emulsifying an aqueous solution directly into the lipidcontrast medium, Lipiodol.

[0065] Another important antineoplastic agent is paclitaxel (i.e.taxol), a member of the class of compounds known as taxines, comes fromthe bark of the Pacific yew tree, Taxus brevifolia. For example, taxolhas been used in treating ovarian, breast, non-small cell lung, and headand neck carcinomas. One of the difficulties in administering taxol isthat the drug is insoluble in water. The present state of the art intaxol formulation requires a 50:50 mixture of Cremophor-EL surfactant(polyoxyethylated castor oil) and ethanol in order to solubilize thedrug. Unfortunately, this taxol formulation leads to a relatively highincidence of major hypersensitivity reactions (HSRs) upon intravenousadministration. These HSRs have been attributed to the unusually highconcentration of Cremophor-EL required to solubilize the taxol.

[0066] There have been other attempts to provide a taxol formulation,the most successful of which has been incorporation of the drug into aliposomal formulation that must be freeze dried and reconstituted priorto use.

[0067] Attempts to formulate taxol in a stable lipid emulsion have beenunsuccessful. Taxol is reported to be insoluble in lipid emulsions suchas Intralipid.®, which contains soybean oil, or Liposyn.®, whichcontains a mixture of soybean and safflower oils. L. C. Collins-Gold etal., “Parenteral Emulsions for Drug Delivery”, Advanced Drug DeliveryReviews, 5, pp. 189-208 (1990). Heating taxol in either soybean oil orsafflower oil, even upon sonication, does not result in the dissolutionof appreciable amounts of taxol, and addition of taxol to a lipidemulsion during the homogenization step meets with equally disappointingresults. Emulsions incorporating up to 15 mg/ml of taxol have beenformulated with triacetin, L-.alpha.-lecithin, Polysorbate 80, PluronicF-68, ethyloleate and glycerol. However, these emulsions are highlytoxic and unstable. B. Tarr et al., “A New Parenteral Emulsion for theAdministration of Taxol”, Pharmaceutical Research, 4, pp. 162-165(1987).

[0068] Kaufman et al (U.S. Pat. No. 5,785,950) have recently overcomethese issues and formulated an oil-in-water emulsion composed of taxine,an oil, water and a surfactant. In this formulation, a taxine such astaxol is solubilized in the oil in an effective pharmaceutical amountfor intravenous administration. The taxine and oil mixture forms adispersed phase in the water. The oil may be any of a number of oilssuch as mineral, vegetable, animal, essential and synthetic oils, ormixtures thereof. Preferably, the oil is rich in triglycerides, such assafflower oil, soybean oil or mixtures thereof. Because taxol is moresoluble in safflower oil than soybean oil, safflower oil is mostpreferred. The surfactant used may be any of a number of surfactants,and usually is a phospholipid such as lecithin. Typically, the taxine ispresent in an amount of about 0.1% to about 1% by weight of theemulsion, while the oil is present in an amount of from about 1% toabout 40% and the surfactant is present in an amount of about 0.5% toabout 5% by weight of the emulsion.

EXAMPLE 1 Preparation of Biotinylated Doxorubicin Emulsion

[0069] Targeting of doxorubicin therapeutic emulsions may be achievedwith a three-step process for “pretargeting” a biotinylated antibody andsubsequent binding of a biotinylated emulsion to a molecular epitope.The emulsion itself is produced by incorporating biotinylatedphosphatidylethanolamine into the outer lipid monolayer of aperfluorocarbon microemulsion. Briefly, the emulsion comprisesperfluorooctylbromide (40% w/v, PFOB, 3M), a surfactant co-mixture(2.0%, w/v) and glycerin (1.7%, w/v). The surfactant co-mixture includes64 mole % lecithin (Pharmacia Inc), 35 mole % cholesterol (SigmaChemical Co.) and 1 mole %N-(6-(biotinoyl)amino)hexanoyl)-dipalmitoyl-L-alpha-phosphatidyl-ethanolamine,Pierce Inc.) which are dissolved in chloroform. Doxorubicin is suspendedin methanol (˜25 μg/20 μl) and added in titrated amounts between 0.01and 5.0 mol % of the 2% surfactant layer, preferably between 0.2 and 2.0mol %, The chloroform-lipid mixture is evaporated under reducedpressure, dried in a 50° C. vacuum oven overnight and dispersed intowater by sonication. The suspension is transferred into a blender cup(Dynamics Corporation of America) with perfluorooctylbromide anddistilled, deionized water and emulsified for 30 to 60 seconds. Theemulsified mixture is transferred to a Microfluidics emulsifier(Microfluidics Co.) and continuously processed at 20,000 PSI for threeminutes. The completed emulsion is vialed, blanketed with nitrogen andsealed with stopper crimp seal until use. A control emulsion can beprepared identically excluding doxorubicin from the surfactantcomixture. Particle sizes are determined in triplicate at 37° C. with alaser light scattering submicron particle size analyzer (MalvernZetasizer 4, Malvern Instruments Ltd, Southborough, Mass.)., whichindicate tight and highly reproducible size distribution with averagediameters less than 400 nm. Unincorporated drug can be removed bydialysis or ultrafiltration techniques.

[0070] Antibody of F_((ab)) fragment prepared as descrbed below isbiotinylated using the EZ-link™ Sulfo-NHS-LC-Biotinylation Kit. Briefly,2 to 10 mg of protein in 1 ml of phosphate buffered saline is combinedwith Sulfo-NHS-LC-Biotin in distilled or deionized water to afford a 12to 20-fold molar excess of reagent to protein. Incubate solution in cefor 2 hours or at room temperature for 30 minutes. Separate biotinylatedprotein from reagents using a 10 ml desalting column equilibrated andeluted with phosphate buffered saline. Collect fractions of eluate andmeasure UV absorbance at 280 nm with a spectrophotometer. Store at 4° C.until use.

EXAMPLE 2 Preparation of Antibody [F_((ab))]-Conjugated DoxorubicinEmulsion

[0071] The perfluorocarbon nanoparticle contrast agent is produced byincorporating 1,2-dipalmitoyl-snglycero-3-phosphoethanolamine-N-4-(p-maleimidophenyl)butyramide (MPB-PE)into the outer lipid monolayer of the emulsion to accommodate ligandconjugation. The emulsion is comprised of perfluorooctylbromide (40%w/v), a surfactant co-mixture (2% w/v), glycerin (1.2% w/v) and water(54.8% w/v). The surfactant co-mixture included lecithin (67.9 mol %),cholesterol (30 mol %), dipalmitoyl-phosphatidylethanolamine (2 mol %)and MPB-PE (0.1 mol %) and is dissolved in chloroform. Doxorubicin issuspended in methanol (˜25 μg/20 μl) and added in titrated amountsbetween 0.01 and 5.0 mol % of the 2% surfactant layer, preferablybetween 0.2 and 2.0 mol %. The chloroform-lipid mixture is evaporatedunder reduced pressure, dried in a 50° C. vacuum oven overnight anddispersed into water by sonication. The suspension is transferred into ablender cup with perfluorocarbon, safflower oil and distilled, deionizedwater and emulsified as above. A control emulsion is preparedidentically except a nonderivatized phosphatidylethanolamine may besubstituted into the surfactant co-mixture. The completed emulsion isvialed, blanketed with nitrogen and sealed with stopper crimp seal untiluse. A control emulsion can be prepared identically excludingdoxorubicin from the surfactant comixture. Particle sizes are determinedin triplicate at 37° C. with a laser light scattering submicron particlesize analyzer (Malvern Zetasizer 4, Malvern Instruments Ltd,Southborough, Mass.)., which indicate tight and highly reproducible sizedistribution with average diameters less than 400 nm. Unincorporateddrug is removed by dialysis.

[0072] F(ab)′ fragments are generated and isolated using an immunopureF(ab)′ preparation kit (Pierce, Rockford, Ill.). Briefly, IgG isdialyzed into 20 mM phosphate/10 mM EDTA buffer (pH 7.0), concentratedto 20 mg/ml and digested by immobilized papain. Solubilized F(ab)′ ispurified from Fc fragments and undigested IgG protein using a protein Acolumn. F(ab)′ fragments is purified from excess cysteine using aG25-150 column and deoxygenated phosphate buffer (pH 6.7). Fractionidentity is confirmed by routine SDS-PAGE procedures. An analogousemulsion using an irrelevant IgG is used to prepare control ligands withrandom specificities.

[0073] F(ab)′ fractions are pooled and combined with the MPB-PEderivatized emulsion (1-2 mg F(ab)′/ml of emulsion). The mixture isadjusted to pH 6.7, sealed under nitrogen and allowed to react overnightat ambient temperatures with gentle, continuous mixing. The mixture issubsequently dialyzed with a 300,000 MWCO Spectra/Por DispoDialyzer(Laguna Hills, Calif.) against 10 mM phosphate buffer (pH 7.2) to removeunconjugated F(ab)′ fragments. The final emulsion is vialed undernitrogen and stored at 4° C. until use. A nonspecific control emulsionmay be prepared using the control, irrelevant IgG F(ab)′ fragments inthe above protocol.

EXAMPLE 3 Effectiveness of Therapeutic Doxorubicin (DXR) NanoparticleEmulsions Targeted to Tissue Factor on Porcine Aortic Smooth MuscleCells

[0074] Example 3 demonstrates the enhanced efficacy of ligand-targeteddoxorubicin nanoparticle emulsions versus free doxorubicon to inhibitthe proliferation of aortic smooth muscle cells in vitro. Pig aorticsmooth muscle cells are seeded onto 12 mm round glass coverslips in 24well cluster plates at a density of 5×10⁴ cells per well (n=45;9/treatment). The cells are grown in smooth muscle basal mediumcontaining 5% FBS for 72 hours, then rinsed in media and incubated on aplatform shaker at 37° C., 95%/5% O/CO2 with medium containingbiotinylated anti-tissue factor antibody (25 μg) for 1 hour. Excessantibody is rinsed from cultures 3×. Cells are next incubated with 25 μgof avidin (Pierce, Rockford, Ill.) for 30 min then washed 3× to removeexcess avidin. Finally, doxorubicin emulsion (25 μl) incorporating 0,0.2 or 2.0 mol % drug within the phospholipid surfactant, is incubatedwith the cells for 30 minutes at 37° C. The cells were washed free ofunbound emulsion or drug 3× and allowed to continue growing at 37° C.,95%/5% O/CO2. After 48 hours additional growth, the cells were dispersedfrom the cover slips with trypsin and counted with a hemacytometer (FIG.1).

[0075] Tissue factor-targeted DXR nanoparticles had greateranti-proliferative effects than the free doxorubicin and thisdifferential effect was greater at the lower dosage of DXR. DXRnanoparticles decreased cell proliferation in a dose responsive manner.The results suggest that DXR formulated into the lipid surfactant wasretained and slowly delivered from the nanoparticles into the smoothmuscle cells. The delivery of DXR into the pig aortic smooth musclecells is facilitated by the intimate, prolonged contact of the emulsionnanoparticles with the cell membrane surface and exchange of lipids anddrug between the two lipid layers.

EXAMPLE 4 Effectiveness of Therapeutic Paclitaxel (Taxol) NanoparticleEmulsions Targeted to Tissue Factor on Porcine Aortic Smooth MuscleCells

[0076] Example 4 demonstrates the efficacy of ligand-targeted paclitaxelnanoparticle emulsions versus a targeted-control emulsion to inhibit theproliferation of aortic smooth muscle cells in vitro. Since paclitaxelis highly insoluble in buffer, comparisons of nanoparticle formulationswith free drug were not possible. Pig aortic smooth muscle cells wereseeded onto 12 mm round glass cover slips in 24 well cluster plates at adensity of 5×10⁴ cells per well (n=18; 6/treatment). The cells weregrown in smooth muscle basal medium containing 5% FBS for 72 hours, thenrinsed in media and incubated on a platform shaker at 37° C., 95%/5%O/CO2 with medium containing biotinylated anti-tissue factor antibody(25 μg) for 1 hour. Excess antibody was rinsed from cultures 3×. Cellswere next incubated with 25 μg of avidin (Pierce, Rockford, Ill.) for 30min then washed 3× to remove excess avidin. Finally, paclitaxelemulsions (25 μl) at concentrations of 0, 0.2 and 2.0 mol % within thephospholipid surfactant, were incubated with the cell cultures for 30minutes at 37° C. The cells were washed free of unbound emulsion (i.e. 3rinses) and allowed to grow at 37° C., 95%/5% O/CO2. After 48 hours, thecells were trypsinized and counted with a hemacytometer (FIG. 2).

[0077] Tissue factor-targeted paclitaxel nanoparticles had marked,equivalent anti-proliferative effects at both dosages of paclitaxel.Paclitaxel, although highly insoluble in aqueous culture medium, waseffectively delivered and inhibited the proliferation of aortic smoothmuscle cells. These results suggest that paclitaxel formulated into thelipid surfactant was delivered from the nanoparticles directly into thesmooth muscle cell membrane. The delivery of paclitaxel to the pigaortic smooth muscle cells was facilitated by the intimate, prolongedcontact of the emulsion nanoparticles with the cell membrane surface andexchange of lipids and drug between the two lipid layers.

EXAMPLE 5 Enhanced Effectiveness of Therapeutic Doxorubicin orPaclitaxel Nanoparticle Emulsions Targeted to Tissue Factor on PorcineAortic Smooth Muscle Cells

[0078] Example 5 demonstrates the enhanced effectiveness ofligand-targeted doxorubicin or paclitaxel nanoparticle emulsions versusnon-targeted nanoparticle emulsion. This example illustrates theimportance of ligand-targeting versus free emulsions. Pig aortic smoothmuscle cells were seeded onto 12 mm round glass cover slips in 24 wellcluster plates at a density of 5×10⁴ cells per well (n=30; 3/treatment).The cells were grown in smooth muscle basal medium containing 5% FBS for72 hours, then rinsed in media and incubated on a platform shaker at 37°C., 95%/5% O/CO2 with medium. One half of the wells receivedbiotinylated anti-tissue factor antibody (25 μg) for 1 hour. Excessantibody and media was rinsed from all cultures 3×. One half of thecells were next incubated with 25 μg of avidin (Pierce, Rockford, Ill.)for 30 min then washed 3× to remove excess avidin and media. Finally,paclitaxel, doxorubicin or control (no drug) emulsions (25 μl) at drugconcentrations of 0, 0.2 and 2.0 mol % within the phospholipidsurfactant, were incubated with the cell cultures for 30 minutes at 37°C. The cells were washed free of unbound emulsion (i.e. 3 rinses) andallowed to grow at 37° C., 95%/5% O/CO2. After 48 hours, the cells weretrypsinized and counted with a hemacytometer (Table 1). TABLE 1 Theeffect of targeted and free therapeutic nanoparticles on porcine aorticcell proliferation (cell counts × 10,000) in vitro. DoxorubicinPaclitaxel (DXR) (Taxol) Antibody Control 0.2 mol % 2.0 mol % 0.2 mol %2.0 mol % Free 23.3 ± 1.8^(a) 12.7 ± 1.0^(b) 7.9 ± 0.3^(c) 25.0 ±1.5^(a) 23.0 ± 1.5^(a) Targeted 27.0 ± 1.2^(a)   6.0 ± 1.9^(c)   1.9 ±0.7^(d)   7.0 ± 0.6^(c)  5.2 ± 0.4^(c)

[0079] Tissue factor-targeted nanoparticles had greateranti-proliferative effects at both dosages of paclitaxel or doxorubicinthan free therapeutic emulsion. The greatest benefit was measured forpaclitaxel, the most hydrophobic compound. The control emulsions, freeor targeted did not inhibit cell proliferation. Free paclitaxel emulsionnanoparticles did not affect cell growth regardless of dosage. Freedoxorubicin emulsion particles did inhibit cell growth in a dose relatedfashion, but the effect was most pronounced when the particles wereactively targeted to the cell surface with anti-tissue factor antibody,an epitope constitutively expressed by these cells. The delivery ofpaclitaxel or doxorubicin into the pig aortic smooth muscle cells wasfacilitated by the intimate, prolonged contact of the emulsionnanoparticles with the cell membrane surface and the exchange of lipidsand drug between the two lipid layers (see FIG. 3).

[0080] As various changes could be made in the above methods andcompositions without departing from the scope of the invention, it isintended that all matter contained in the above description and shown inthe accompanying drawings shall be interpreted as illustrative and notin a limiting sense.

What is claimed is:
 1. A composition for use in delivering a bioactiveagent to targeted tissues or cells comprising: (a) a site-specifictargeting ligand; (b) a lipid encapsulated oil in water emulsion; and(c) a bioactive agent in or on the surface of the outer monolayer saidemulsion; said ligand being conjugated directly or indirectly to saidemulsion and said composition providing facilitated delivery of saidbioactive agent through prolonged association and increased contact ofthe ligand-bound, lipid encapsulated emulsion particles with the lipidbilayer of said target tissues or cells.
 2. A composition as set forthin claim 1 wherein said site-specific targeting ligand is selected fromthe group consisting of antibodies, antibody fragments, peptides,asialoglycoproteins, polysaccharides, aptamers, small molecules, nucleicacids, peptidomimetics, mimetics and drugs.
 3. A composition as setforth in claim 2 wherein said site-specific targeting ligand is anantibody.
 4. A composition as set forth in claim 1 wherein said oil inwater emulsion contains a fluorochemical.
 5. A composition as set forthin claim 4 wherein said fluorochemical is a fluorocarbon.
 6. Acomposition as set forth in claim 4 wherein said fluorocarbon isperfluorooctylbromide.
 7. A composition as set forth in claim 4 whereinsaid fluorochemical is a liquid with a boiling point above approximately30° C.
 8. A composition as set forth in claim 7 wherein saidfluorochemical liquid has a boiling point above approximately 90° C. 9.A composition as set forth in claim 1 wherein said bioactive agent isselected from the group consisting of chemotherapeutic agents, drugs,genetic materials, nucleic acid-based therapy, protein or peptidetherapy, radioactive isotopes or combinations thereof.
 10. A compositionas set forth in claim 9 wherein said bioactive agent is achemotherapeutic agent.
 11. A composition as set forth in claim 1wherein said lipid encapsulated emulsion has an outer coating or outermonolayer composed of a material selected from the group consisting of anatural or synthetic phospholipid, a fatty acid, cholesterol, lysolipid,sphingomyelin, tocopherol, glucolipid, stearylamine, cardiolipin, alipid with ether or ester linked fatty acids and a polymerized lipid.12. A composition as set forth in claim 1 wherein said outer monolayerof said emulsion also contains an additional surfactant incorporatedtherein for stabilizing said emulsion.
 13. A composition as set forth inclaim 12 wherein said additional surfactant is selected from the groupconsisting of nonionic and amphoteric surfactants.
 14. A composition asset forth in claim 12 wherein said surfactant contains a cationic lipidto facilitate adhesion of said bioactive agent to said emulsionparticles.
 15. A composition as set forth in claim 14 wherein saidcationic lipid is selected from the group consisting ofN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride,1,2-dioleoyloxy-3-(trimethylammonio)propane,1,2-dioleoyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol,1,2-diacyl-3-trimethylammonium-propane,1,2-diacyl-3-dimethylammonium-propane,1,2-diacyl-sn-glycerol-3-ethylphosphochloine, and3β-[N′,N′-dimethylaminoethane)-carbamol]cholesterol-HCl.
 16. Acomposition as set forth in claim 1 wherein said emulsion contains anemulsifying and/or solubilizing agent.
 17. A composition as set forth inclaim 1 wherein said emulsion particles have a diameter in the range ofapproximately 0.01 to 10 microns.
 18. A composition as set forth inclaim 17 wherein said emulsion particles have a diameter in the range ofapproximately 0.1 to 0.5 microns.
 19. A composition as set forth inclaim 1 wherein said emulsion contains a primer material incorporatedinto the outer monolayer thereof to covalently bond said ligand withsaid emulsion.
 20. A composition as set forth in claim 19 wherein saidprimer material is selected from the group consisting of1-ethyl-3-(3-N-N-dimethylaminopropyl)carbodiimide hydrochloride,1-cyclohexyl-3-(2-morpholinoethyl)carbodimide methyl-p-toluenesulfonate,phosphatidylethanolamine, N-caproylamine phosphatidylethanolamine,N-dodecanylamine phosphatidylethanolamine, phosphotidylthioethanol,1,2-diacyl-sn-glycerol-3-phosphoethanolamine-N-[4-p-maleimidephenyl)-butyramide,N-succinyl-phosphatidylethanolamine,N-glutaryl-phosphatidylethanolamine,N-dodecanyl-phosphatidylethanolamine,N-biotinyl-phosphatidylethanolamine,N-biotinylcaproyl-phosphatidylethanolamine, and phosphatidylethyleneglycol.
 21. A composition as set forth in claim 1 wherein said ligand isconjugated directly to said emulsion by direct adsorption of said ligandto the oil/aqueous interface of said emulsion.
 22. A composition for usein delivering a bioactive agent to targeted tissues or cells comprising:(a) a lipid encapsulated oil in water emulsion; and (b) a combinationsite-specific targeting ligand/bioactive agent in or on the surface ofthe outer monolayer of said emulsion; said combination ligand/bioactiveagent being conjugated directly or indirectly to said emulsion and saidcomposition providing facilitated delivery of said bioactive agentthrough prolonged association and increased contact of the ligand-bound,lipid encapsulated emulsion particles with the lipid bilayer of saidtarget tissues or cells.
 23. A composition as set forth in claim 22wherein said combination ligand/bioactive agent is selected from thegroup consisting of antibodies, peptide fragments and mimetics.
 24. Acomposition as set forth in claim 23 wherein said combinationligand/bioactive agent is an antibody.
 25. A composition as set forth inclaim 22 wherein said oil in water emulsion contains a fluorochemical.26. A composition as set forth in claim 25 wherein said fluorochemicalis a fluorocarbon.
 27. A composition as set forth in claim 25 whereinsaid fluorochemical is a liquid with a boiling point above approximately30° C.
 28. A composition as set forth in claim 27 wherein saidfluorochemical liquid has a boiling point above approximately 90° C. 29.A composition as set forth in claim 22 wherein said lipid encapsulatedemulsion has an outer coating or outer monolayer composed of a materialselected form the group consisting of a natural or syntheticphospholipid, a fatty acid, cholesterol, lysolipid, sphingomyelin,tocopherol, glucolipid, stearylamine, cardiolipin, a lipid with ether orester linked fatty acids and a polymerized lipid.
 30. A composition asset forth in claim 22 wherein said outer monolayer of said emulsion alsocontains an additional surfactant incorporated therein for stabilizingsaid emulsion.
 31. A composition as set forth in claim 30 wherein saidsurfactant contains a cationic lipid to facilitate adhesion of saidligand/bioactive agent to said emulsion particles.
 32. A composition asset forth in claim 22 wherein said emulsion contains an emulsifyingagent and/or solubilizing agent.
 33. A composition as set forth in claim27 wherein said emulsion particles have a diameter in the range ofapproximately 0.01 to 10 microns.
 34. A composition as set forth inclaim 33 wherein said emulsion particles have a diameter in the range ofapproximately 0.1 to 0.5 microns.
 35. A composition as set forth inclaim 22 wherein said emulsion contains a primer material incorporatedinto the outer lipid monolayer thereof to covalently bond saidligand/bioactive agent with said emulsion.
 36. A composition as setforth in claim 22 wherein said ligand/bioactive agent is conjugateddirectly to said emulsion by direct adsorption of the ligand/bioactiveagent to the oil/aqueous interface of said emulsion.
 37. A method forimproved delivery of a bioactive agent to targeted tissues or cellscomprising administering to said tissues or cells a compositioncomprising: (a) a site-specific targeting ligand; (b) a lipidencapsulated oil in water emulsion; and (c) a bioactive agent in or onthe surface of the outer monolayer of said emulsion; said ligand beingconjugated directly or indirectly to said emulsion; whereby saidcomposition provides facilitated delivery of said bioactive agentthrough prolonged association and increased contact of the ligand-bound,lipid encapsulated emulsion particles with the lipid bilayer of saidtarget tissues or cells.
 38. A method as set forth in claim 37 whereinsaid site-specific targeting ligand is selected from the groupconsisting of antibodies, antibody fragments, peptides,asialoglycoproteins, polysaccharides, aptamers, small molecules, nucleicacids, peptidomimetics, mimetics and drugs.
 39. A method as set forth inclaim 38 wherein said site-specific targeting ligand is an antibody. 40.A method as set forth in claim 37 wherein said oil in water emulsioncontains a fluorochemical.
 41. A method as set forth in claim 40 whereinsaid fluorochemical is a fluorocarbon.
 42. A method as set forth inclaim 41 wherein said fluorocarbon is perfluorooctylbromide.
 43. Amethod as set forth in claim 40 wherein said fluorochemical is a liquidwith a boiling point above approximately 30° C.
 44. A method as setforth in claim 43 wherein said fluorochemical liquid has a boiling pointabove approximately 90° C.
 45. A method as set forth in claim 37 whereinsaid bioactive agent is selected from the group consisting ofchemotherapeutic agents, drugs, genetic materials, nucleic acid-basedtherapy, protein or peptide therapy, radioactive isotopes orcombinations thereof.
 46. A method as set forth in claim 45 wherein saidbioactive agent is a chemotherapeutic agent.
 47. A method as set forthin claim 45 wherein said lipid encapsulated emulsion has an outercoating composed of a material selected from the group consisting of anatural or synthetic phospholipid, a fatty acid, cholesterol, lysolipid,sphingomyelin, tocopherol, glucolipid, stearylamine, cardiolipin, alipid with ether or ester linked fatty acids and a polymerized lipid.48. A method as set forth in claim 33 wherein said outer monolayer ofsaid emulsion also contains an additional surfactant incorporatedthereon for stabilizing said emulsion.
 49. A method as set forth inclaim 48 wherein said additional surfactant is selected from the groupconsisting of nonionic and amphoteric surfactants.
 50. A method as setforth in claim 37 wherein said surfactant contains a cationic lipid tofacilitate adhesion of said bioactive agent to said emulsion particles.51. A method as set forth in claim 37 wherein said emulsion contains anemulsifying and/or solubilizing agent.
 52. A method as set forth inclaim 37 wherein said emulsion particles have a diameter in the range ofapproximately 0.01 to 10 microns.
 53. A method as set forth in claim 52wherein said emulsion particles have a diameter in the range ofapproximately 0.1 to 0.5 microns.
 54. A method as set forth in claim 37wherein said emulsion contains a primer material incorporated into theouter monolayer thereof to covalently bond said ligand with saidemulsion.
 55. A method as set forth in claim 37 wherein said ligand isconjugated directly to said emulsion by direct adsorption of said ligandto the oil/aqueous outerface of said emulsion.
 56. A method for improveddelivery of a bioactive agent to targeted tissues or cells comprisingadministering to said tissues or cells a composition comprising: (a) alipid encapsulated oil in water emulsion; and (b) a combinationsite-specific targeting ligand/bioactive agent in or on the surface ofthe outer monolayer of said emulsion; said combination ligand/bioactiveagent being conjugated directly or indirectly to said emulsion and saidcomposition providing facilitate delivery of said bioactive agentthrough prolonged association and increased contact of the ligand-bound,lipid encapsulated emulsion particles with the lipid bilayer of saidtarget tissues or cells.
 57. A method as set forth in claim 50 whereinsaid combination ligand/bioactive agent is selected from the groupconsisting of antibodies, antibody fragments, peptides,asialoglycoproteins, polysaccharides, aptamers, small molecules, nucleicacids, peptidomimetics, mimetics and drugs.
 58. A method as set forth inclaim 57 wherein said combination ligand/bioactive agent is an antibody.59. A method as set forth in claim 56 wherein said oil in water emulsioncontains a fluorochemical.
 60. A method as set forth in claim 59 whereinsaid fluorochemical is a fluorocarbon.
 61. A method as set forth inclaim 59 wherein said fluorochemical is a liquid with a boiling pointabove approximately 30° C.
 62. A method as set forth in claim 61 whereinsaid fluorochemical liquid has a boiling point above approximately 90°C.
 63. A method as set forth in claim 56 wherein said lipid encapsulatedemulsion has an outer coating or outer monolayer composed of a materialselected from the group consisting of a natural or syntheticphospholipid, a fatty acid, cholesterol, lipolipid, sphengomyelin,tocopheral, glucolipid, stearylamine, cardiolipin, a lipid with ether orester linked fatty acids and a polymerized lipid.
 64. A method as setforth in claim 56 wherein said outer monolayer of said emulsion alsocontains an additional surfactant incorporated therein for stabilizingsaid emulsion.
 65. A method as set forth in claim 64 wherein saidsurfactant contains a cationic lipid to facilitate adhesion of saidligand/bioactive agent to said emulsion particles.
 66. A method as setforth in claim 56 wherein said emulsion contains an emulsifying agentand/or solubilizing agent.
 67. A method as set forth in claim 56 whereinsaid emulsion particles have a diameter in the range of approximately0.01 to 10 microns.
 68. A method as set forth in claim 67 wherein saidemulsion particles have a diameter in the range of approximately 0.1 to0.5 microns.
 69. A method as set forth in claim 56 wherein said emulsioncontains a primer material incorporated into the outer lipid monolayerthereof to covalently bond said ligand/bioactive agent with saidemulsion.
 70. A method as set forth in claim 56 wherein saidligand/bioactive agent is conjugated directly to said emulsion by directadsorption of the ligand/bioactive agent to the oil-aqueous interface ofsaid emulsion.